Periodic Reporting for period 4 - INSITE (Development and use of an integrated in silico-in vitro mesofluidics system for tissue engineering)
Reporting period: 2023-03-01 to 2023-08-31
Tissue Engineering (TE) refers to the branch of medicine that aims to replace or regenerate functional tissue or organs using man-made living implants. These living implants, unlike implants from metal or plastic, can integrate perfectly in the body and, in case of children, can grow as the patient grows. Tissue Engineering is part of the solution to tackle the world-wide donor organ and tissue shortage.
As the field is moving towards more complex TE constructs with sophisticated functionalities, there is a lack of dedicated in vitro devices that allow testing the response of the complex construct as a whole, prior to implantation. This means that the field heavily relies on animal experimentation, which has many well-documented limitations. Additionally, the knowledge accumulated from mechanistic and empirical in vitro and in vivo studies is often underused in the development of novel constructs due to a lack of integration of all the data in a single, in silico, platform.
The INSITE project aimed to address both challenges by developing a new fluidics set-ups for in vitro testing of TE constructs and by developing dedicated multiscale and multiphysics models that aggregate the available data and use these to design complex constructs and proper fluidics settings for in vitro testing. The combination of these in silico and in vitro approaches will lead to an integrated knowledge-rich in silico-in vitro system that provides an in vivo-like time-varying environment. The system will emulate the in vivo environment present at the (early) stages of bone regeneration including the vascularization process and the innate immune response. A proof of concept will be delivered for complex TE constructs for large bone defects and and non-healing fractures.
As the field is moving towards more complex TE constructs with sophisticated functionalities, there is a lack of dedicated in vitro devices that allow testing the response of the complex construct as a whole, prior to implantation. This means that the field heavily relies on animal experimentation, which has many well-documented limitations. Additionally, the knowledge accumulated from mechanistic and empirical in vitro and in vivo studies is often underused in the development of novel constructs due to a lack of integration of all the data in a single, in silico, platform.
The INSITE project aimed to address both challenges by developing a new fluidics set-ups for in vitro testing of TE constructs and by developing dedicated multiscale and multiphysics models that aggregate the available data and use these to design complex constructs and proper fluidics settings for in vitro testing. The combination of these in silico and in vitro approaches will lead to an integrated knowledge-rich in silico-in vitro system that provides an in vivo-like time-varying environment. The system will emulate the in vivo environment present at the (early) stages of bone regeneration including the vascularization process and the innate immune response. A proof of concept will be delivered for complex TE constructs for large bone defects and and non-healing fractures.
One part of the INSITE project focused on developing a multi-omics workflow and a mechanistic model of the in vitro biology observed in the fluidic device. This included the development of single-cell sequencing analysis pipelines and the creation of a comprehensive bone development and homeostasis atlas based on scRNASeq datasets. An app accompanying this atlas has been developed for community use. Metabolomics studies on human Periosteum Derived Cells (hPDCs) identified several indicators for successful in vivo bone regeneration. An intracellular network model was established, resulting in two shareable outputs: a knowledge base in CellCollective and an executable chondrocyte model for external use. Additionally, tissue-level neotissue growth models were recalibrated for different substrates and integrated into a multiscale model.
A second part of the project centered on developing the INSITE fluidic device, resulting in a fully sensored prototype with inline pH, oxygen, and temperature sensors. Neotissue growth was studied in 3D printed calcium phosphate scaffolds, and a second microfluidics device was optimized to apply mechanical loading to cells in hydrogels. This device was used to examine the effects of mechanical loading on macrophages, chondrocytes, and osteoprogenitor cells, providing input for other parts of the work in INSITE.
A third part of the project involved analyzing early bone healing phases and emulating the biophysical context within the in vitro system. Computational models of vascularization and immune response were developed, including an agent-based inflammatory model, validated through dedicated experiments. Single-cell RNA sequencing experiments are currently being analyzed to quantify vascularization and early immune responses during fracture healing under various mechanical loading regimes.
Finally, puttiing together all the aformentioned elements, we developed spatio-temporally patterned constructs for bone defect healing, utilizing a high-end bioprinter to print 3D cell-laden constructs. Various hydrogel blends were developed to enhance printability and mechanical properties. In vivo experiments with these printed implants are ongoing. Biomaterials-based constructs were also optimized and are being prepared for translation to clinical use.
Overall, the technologies and tools developed have broad applications beyond the initial scope, including lymphangiogenesis studies in inflammatory conditions and multi-organ toxicity applications. This work has been presented at conferences and is in various stages of publication.
A second part of the project centered on developing the INSITE fluidic device, resulting in a fully sensored prototype with inline pH, oxygen, and temperature sensors. Neotissue growth was studied in 3D printed calcium phosphate scaffolds, and a second microfluidics device was optimized to apply mechanical loading to cells in hydrogels. This device was used to examine the effects of mechanical loading on macrophages, chondrocytes, and osteoprogenitor cells, providing input for other parts of the work in INSITE.
A third part of the project involved analyzing early bone healing phases and emulating the biophysical context within the in vitro system. Computational models of vascularization and immune response were developed, including an agent-based inflammatory model, validated through dedicated experiments. Single-cell RNA sequencing experiments are currently being analyzed to quantify vascularization and early immune responses during fracture healing under various mechanical loading regimes.
Finally, puttiing together all the aformentioned elements, we developed spatio-temporally patterned constructs for bone defect healing, utilizing a high-end bioprinter to print 3D cell-laden constructs. Various hydrogel blends were developed to enhance printability and mechanical properties. In vivo experiments with these printed implants are ongoing. Biomaterials-based constructs were also optimized and are being prepared for translation to clinical use.
Overall, the technologies and tools developed have broad applications beyond the initial scope, including lymphangiogenesis studies in inflammatory conditions and multi-organ toxicity applications. This work has been presented at conferences and is in various stages of publication.
The INSITE project has allowed to advance work in several scientific domains related to regenerative medicine and in silico modeling. Key results include the following.
- Skeletal cell atlas: the Human Cell Atlas initiative has produced many atlases, yet the musculoskeletal one is still missing. In the INSITE project, we developed the limb skeletal cell atlas. It was reviewed, and accepted, for methodological accuracy by specialists. The impact of the atlas will be measured by the uptake by the community. At conferences, the community has shown a particular interest in the tool and wants to use it as a bench mark for their own studies.
- Model-based scaffold optimization: a tissue growth model has been developed for different materials types (e.g. Calcium Phosphate) and used to identify interesting scaffold shapes that allow for easy additive manufacturing as well as sufficient space for neotissue ingrowth. A range of in vitro and in vivo experiments, in collaboration with clinical and industrial partners, allowed for model validation. Clinical translation is ongoing for pediatric patients with large non-union bone defects.
- Fluidic tools: several tools have been optimized for the in vitro assessment of biological phenomena under different environmental conditions. These results are used both as input for model building, calibration and validation, as well as for the evaluation of the in vivo potential of the designed tissue engineered constructs.
The work in the INSITE project furthermore allowed to build up collaborations with a range of different companies and research centers, including biomaterials, 3D printing and microfluidics companies. In these collaborations, tools developed by the INSITE team are explored for use in different settings. Finally, the INSITE project and the results obtained, provided opportunities to engage with policy makers in Belgium and at the European level. This has resulted in several initiatives that aim to bring together the digital twin ecosystem and to develop a shared vision and roadmap for the future implementation of the Virtual Human Twin program.
- Skeletal cell atlas: the Human Cell Atlas initiative has produced many atlases, yet the musculoskeletal one is still missing. In the INSITE project, we developed the limb skeletal cell atlas. It was reviewed, and accepted, for methodological accuracy by specialists. The impact of the atlas will be measured by the uptake by the community. At conferences, the community has shown a particular interest in the tool and wants to use it as a bench mark for their own studies.
- Model-based scaffold optimization: a tissue growth model has been developed for different materials types (e.g. Calcium Phosphate) and used to identify interesting scaffold shapes that allow for easy additive manufacturing as well as sufficient space for neotissue ingrowth. A range of in vitro and in vivo experiments, in collaboration with clinical and industrial partners, allowed for model validation. Clinical translation is ongoing for pediatric patients with large non-union bone defects.
- Fluidic tools: several tools have been optimized for the in vitro assessment of biological phenomena under different environmental conditions. These results are used both as input for model building, calibration and validation, as well as for the evaluation of the in vivo potential of the designed tissue engineered constructs.
The work in the INSITE project furthermore allowed to build up collaborations with a range of different companies and research centers, including biomaterials, 3D printing and microfluidics companies. In these collaborations, tools developed by the INSITE team are explored for use in different settings. Finally, the INSITE project and the results obtained, provided opportunities to engage with policy makers in Belgium and at the European level. This has resulted in several initiatives that aim to bring together the digital twin ecosystem and to develop a shared vision and roadmap for the future implementation of the Virtual Human Twin program.